Understanding the fetal to adult hemoglobin switch in humans is important, especially given the clear role that elevated levels of fetal hemoglobin (HbF) have in ameliorating the clinical symptoms of sickle cell disease and 2-thalassemia. Work by others identified three transcription factors that regulate 3 globin gene expression, MYB, BCL11A, and KLF1. This grant builds on and extends our recent discoveries concerning the important roles of two highly conserved and cotranscribed miRNAs - miR-15a, miR-16a - in gamma globin gene regulation. In human trisomy 13 there is delayed switching and persistence of fetal hemoglobin (HbF) and elevation of embryonic hemoglobin in newborns. We localized the critical region to chromosomal band 13q14 and identified enhanced expression of miR-15a and 16-1 as top candidates for the elevated HbF levels. Indeed, we showed that a 50% increased expression of these microRNAs in primary human erythroid progenitor cells resulted in elevated fetal and embryonic hemoglobin gene expression. We showed that Myb is one conserved mRNA target of miR-15a and 16-1 in erythroid cells that regulate 3 globin expression. Here we will first determine which erythroid progenitor cell is the primary erythropoietic target of miR-15a- 16-1 by knocking down miR-15a-16-1 expression in purified cultured erythroid progenitors and by analyzing fetal and adult erythropoiesis in mice deleted for the miR15a-16-1 locus. In parallel we will study the effects of modest (50%) overexpression of miR-15- 16 in cultured human and murine erythroid progenitors and also in mice we will generate in which miR-15- 16 is overexpressed by ~50% in all hematopoietic cells or only in erythroid cells. We will determine effects of miR-15a-16- knockdown and modest overexpression on cell cycle and cell proliferation, self renewal of BFU-E progenitors, and on erythroid differentiation, focusing on expression of major erythroid proteins including the human adult, fetal, and embryonic globins. We will also determine the network of mRNAs targeted by miR-15- 16 at different stages of erythropoiesis. Specifically, we will determine the global changes in mRNA levels and translation induced by miR-15a-16-1 overexpression and knockdown in human and murine cells (mRNA- seq) and by analysis of changes in ribosome occupancy (iribosome-footprinting assay). We will then analyze the combined experimental data by several computational approaches to construct a network of miRNA- mRNA interactions that ultimately modify expression of key human and murine erythroid proteins including 3 globin. Using reporter gene assays we will validate key human and murine erythroid mRNAs as targets of miR-15- 16 and then determine the function of key mRNAs as targets by appropriate knockdown or overexpression experiments, focusing on human globin gene regulation. Our overriding goal is thus to identify additional genes that regulate 3 globin gene expression.